人血清白蛋白修饰人粒细胞集落刺激因子和干扰素α-2a
文献类型：学位论文
| 作者 | 朱晓囡 |
| 学位类别 | 硕士 |
| 答辩日期 | 2003 |
| 授予单位 | 中国科学院过程工程研究所 |
| 授予地点 | 中国科学院过程工程研究所 |
| 导师 | 苏志国 |
| 关键词 | 人血清白蛋白 粒细胞集落刺激因子 干扰素 化学修饰 化学偶联 药用蛋白 |
| 其他题名 | Conjugation of Human Serum Albumin with Human Granulocyte Colony Stimulating Factor and Interferon α-2a |
| 学位专业 | 生物化工 |
| 中文摘要 | 用化学修饰方法对蛋白质进行改造具有重要的生物医学意义,可以延长蛋白质药物的循环半衰期,提高药效,降低副作用.其中蛋白质分子之间的化学偶联技术具有较大优越性.该文用异型双功能试剂N-琥珀酰亚胺-6-[3'-(2-吡啶二硫)丙酰胺]已酸酯(N-Succinimidyl-6-[3'-(2-pyridyldithio)propionamido]hexanoate,LC-SPDP)及N-琥珀酰亚胺-3-(2-吡啶二硫)丙酸酯(N-succinimidyl-3-(2-pyridyldithio)-propionate,SPDP)为交联剂,对含自由巯基和不含自由巯基的两类药用蛋白与人血清白蛋白(humanserumalbumin,NSA)的偶联进行了系统研究. |
| 英文摘要 | Chemical modification of proteins is of great significance in biomedical area, in which conjugation of two proteins using chemical cross-linking method is one of the most important technologies. After conjugation, proteins usually have longer circulating half life, better curative effect and fewer side effects. In this thesis, human granulocyte-colony stimulating factor (G-CSF) and interferon a-2a (IFNa-2a) were covalently conjugated to human serum albumin (HSA) by N-Succinimidyl-6- [3'-(2-pyridyldithio) propionamido] hexanoate (LC-SPDP) or N-succinimidyl- 3-(2-pyridyldithio)- propionate (SPDP) in attempt to increase their circulating half life. Conjugation of HSA and G-CSF was first investigated. N-hydroxysuccinimide ester group was used to react with the primary amino group in HSA, followed by reaction between 2-pyridyl-disulfide group and the free sulfhydryl of Cysl7 in G-CSF. By controlling the amount of LC-SPDP, HSA and G-CSF, an HSA-G-CSF conjugate with molecular weight of 105kDa was successfully prepared, in which the molar ratio of HSA to G-CSF is 1:2. The molecular amount of the conjugate was evaluated by HPLC and SDS-PAGE. The unreacted G-CSF was removed by DEAE Sepharose Fast Flow chromatography. NFS-60 cell line was used to measure the activity and the stability of conjugate. It was turned out that the conjugate remained 20% activity of" native G-CSF. However, its heat stability, the stability in serum and the ability to resist protease increased significantly. As for IFN, There is n free sulfhydryl available. Therefore, we took a different approach. IFN was,conjugated to SPDP via NHS-ester group. 2-pyridyl-disulfide group was introduced when reacted with the primary amine group of HSA. The pyridine -2-thione groups were released by incubating with DTT. The free sulfhydryl-containing HSA was then added to the 2-pyridyl disulfide-activated IFN. As a result of the sulfhydryl exchange reaction, HSA and IFN was conjugated. In order to keep the activity of the conjugate, the ratio of SPDP to IFN was controlled below 4. Meanwhile, the influence of reaction pH on IFN activity was studied. It was found, for the first time, that activity is not only related to the extent of NH2 modification, but also to the reaction pH. IFN reacted with SPDP under lower pH showed less activity loss. Therefore, the reaction was carried out in buffer of pH5. The molecular weight of resulted conjugate was evaluated as 85kDa by SDS-PAGE. The in-vitro activity and stability of conjugate were investigated using WISH-VSV system. Although the activity of the conjugate was 25% of the activity of native IFN, the results showed that its heat stability, stability in serum and the anti-protease ability increased remarkably. |
| 语种 | 中文 |
| 公开日期 | 2013-09-16 |
| 页码 | 84 |
| 源URL | [http://ir.ipe.ac.cn/handle/122111/1356]  |
| 专题 | 过程工程研究所_研究所（批量导入） |
| 推荐引用方式 GB/T 7714 | 朱晓囡. 人血清白蛋白修饰人粒细胞集落刺激因子和干扰素α-2a[D]. 中国科学院过程工程研究所. 中国科学院过程工程研究所. 2003. |
入库方式： OAI收割
来源：过程工程研究所
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